Download Benedict`s Test for Reducing Sugars

Applied Biology 2014
Chapter 5: MACROMOLECULES LAB: To determine the identity of unknown
macromolecule solutions and extract DNA from a fruit!
Prelab: Complete this in your lab notebooks. This lab has a different prelab format as it is not an
experiment (so no hypothesis! Or variables..)
Define the purpose:
Background: 1) What are macromolecules?
2) What are 4 types (mmm you know this in your sleep correct?) of macromolecules?
3) Give the monomer and polymer names for each type.
4) What are the reactions that make polymers/macromolecules called?
5) When are these reactions used in the body?
6) What is the purpose of this lab?
7) What are the 4 tests that we shall use in this lab to test for macromolecules and what specific
macromolecules (not your food!) are expected to give positive results for each (read this
handout and figure it out)?
8) Is this a qualitative or quantitative lab in terms of the results collected?
9) What is the difference between a positive and a negative control?
10) Why are the positive/negative controls used in this lab?
11) Once again, read carefully which macromolecules are expected to give positive results for
each test – and complete the table on Pg 4 – complete the ‘PREDICT’ columns for the
controls you are bringing. Ideal positive control will be expected to give a ‘++++’ and
negative control a ‘0’ on the test.
12) Glue down the results data table in your prelab notebook – it is in this hand out on Pg 4.
Bring a positive and negative ‘ideal control’ from home for this lab FOR EACH TEST. Controls
may overlap between tests – you may bring only 5 solutions in all. Food has to be in liquid form –
cannot be water – pl. don’t add stuff to water like salt to ‘technically’ bypass this
requirement!
Your goal in the lab: Find the macromolecules in the four unknown powders/solutions given to
you. Rate them based on the test results for which one has the maximum amount of each type of
macromolecule. Compare the test results from the unknowns to known positive and negative
controls to confirm your results. Use the following tests and run them on the unknowns as well as
your positive and negative controls.
Take one mini spoon of each of the unknown powders and add 10 ml of water – use this
solution to test for Benedict’s (reducing )sugars, Iodine test (polysachcharide) and Biuret’s
(protein). Apply a small pinch of the powder directly on the brown paper test for fats.
A) Benedict's Test for Reducing Sugars
Which sugars will react in this test: The Benedict's test allows us to detect the presence of
monosaccharides (simple sugars). All monosaccharides are reducing sugars; Some disaccharides
like maltose have exposed carbonyl groups and are also reducing sugars (less reactive than
monosaccharides). Other disaccharides such as sucrose are non-reducing sugars and will not react
with Benedict's solution. Starches are also non-reducing sugars.
Applied Biology 2014
Rating Scale: Increasing amounts of reducing simple sugars
blue
green
orange
red
(-)
(+)
(++)
(+++)
reddish brown
(++++)
To Test For Reducing Sugars (monosaccharides and some disaccharides):
1)
2)
3)
4)
5)
Add 2 ml of unknown/control solution
Add 1.5 ml of Benedict’s solution (WEAR GLOVES + GOGGLES!!)
Mix gently. Make sure tube is labelled on the top – as the label will soak in water!
Place test tube in a boiling water bath
Record the color development in 3-5 min using the rating scale above.
B) Starch Test For Polysaccharides
Amylose in polysachcharides is responsible for the formation of a deep blue
color in the presence of iodine. The iodine molecule slips inside of the amylose coil.
To Test For Starch:
1) Add 1 ml of unknown/control in a test tube
2) Add 1 drop of Iodine solution
3) Record color development against a white paper placed behind the test tube
Brown (-)
Blue-Black (++++)
C) Biuret’s Test For Proteins:
The Biuret Reagent is made of sodium hydroxide and copper sulfate. The blue reagent turns violet
in the presence of proteins, and changes to pink when combined with short-chain polypepties. In
this test for proteins there is a reaction between the copper ions and the amino groups in the
peptide bonds.
To Test For Proteins:
1)
2)
3)
4)
Add 2 ml of unknown/control solution to a test tube
Add 4 drops of Biuret’s Reagent (WEAR GLOVES AND GOGGLES!!)
Mix gently
Record color development against a white background
Blue (-)
Slight Purple/Pink (+) (polypeptide)
D) Brown Paper Test for Fats:
Lipids make a translucent spot on a brown paper bag.
Violet (++++)(proteins)
Applied Biology 2014
1)
2)
3)
4)
Rub the unknown/control on a brown paper bag – mark the spot and label.
Dry thoroughly
Hold against light
Record appearance of translucent spot
No spot (-)
Translucent Spot (++++)
E) DNA Spooling – on Thursday! – only performed with the fruit – strawberry or kiwi!
1. Add 2 grams of table salt (NaCl) and 10 ml of cleaning detergent (DAWN) into a 100-ml
measuring cylinder containing 90 ml distilled water. Swell to mix the contents completely.
This is the cell-lysis buffer.
2. Cut your fruit into small slices (about 10 mm thick) and smush it in a Ziploc bag for 2 min.
Add 2 ml of the cell lysis buffer and mush it again for 1 min. Some fruits may need
blending. [Warning: too much blending would shear the DNA molecules.]
3. Filter the slurry from the liquid through a cheese cloth (sitting in a filter funnel) into a 100ml beaker. Each group should collect about 3 ml. [This filtering separates the cell wall
material and protein (remains in the cheese cloth) from DNA, which is now in solution.]
4. Add equal volume (3 ml) of ice-cold ethanol slowly onto the surface of the fruit extract
carefully. [The ethanol must be ice cold - kept in the freezer overnight beforehand.]
5. Immerse a bamboo skewer with a bent tip at the interface of the two layers (alcohol and fruit
layer), stir slowly and continuously in a small circle (clockwise direction) to collect the
DNA thread at the tip of the Pasteur pipette. [DNA doesn't dissolve in ethanol - it comes out
of the lower layer into the upper layer. DNA has the appearance of white mucus.]
Theory: The liquid detergent causes the cell membrane to break down and dissolves the lipids
and proteins of the cell by disrupting the bonds that hold the cell membrane together. The
detergent causes lipids and proteins to precipitate out of the solution. This is filtered out in the
cheese cloth. Next, NaCl enables nucleic acids to precipitate out of an alcohol solution because
it shields the negative phosphate end of DNA, causing the DNA strands to come closer together
and coalesce. Now you know the trick!
Qualitative observations during DNA spooling: record in lab book
Applied Biology 2014
Results from macromolecule lab: ++++ is highest, 0 is lowest. Shaded regions are tests that are
not performed – you may perform them if you wish! Fill in your ratings here:
Unknown/Food
Solution Tested
Benedict’s Test
Starch Test
Biuret’s Test
Fat Test
Predict
Predict
Predict
Predict
Actual
Actual
Unknown 1
Unknown 2
Unknown 3
Unknown 4
Carbohydrate test:
Positive Control
Monosaccharide
Negative Control
Monosaccharide
Positive Control
Polysachcharide
Negative Control
Polysachcharide
Protein test:
Positive Control
Protein
Negative Control
Protein
Lipid Test
Positive Control
Lipid
Negative Control
Lipid
Repeats if any:
Notes on the results/additional observations: in prelab book
Actual
Actual